CN114410618A

CN114410618A - Preparation method of immobilized microorganism carrier, product and application thereof

Info

Publication number: CN114410618A
Application number: CN202111646954.6A
Authority: CN
Inventors: 袁可; 曹娜
Original assignee: Taiyuan University of Technology
Current assignee: Taiyuan University of Technology
Priority date: 2021-12-30
Filing date: 2021-12-30
Publication date: 2022-04-29
Anticipated expiration: 2041-12-30
Also published as: CN114410618B

Abstract

The invention discloses a preparation method of an immobilized microorganism carrier, a product and an application thereof, relating to the technical field of coking wastewater treatment; the method comprises the following steps: adding polyvinyl alcohol, sodium alginate and calcium carbonate into deionized water, and heating and dissolving to be gelatinous to obtain a PVA mixed solution; adding hydrochloric acid into the PVA mixed solution, and reacting to obtain a carrier after hole making; mixing the high-efficiency bacterium Comamonas sp.ZF-3 fermentation thallus, activated sludge, powdered activated carbon and polyhydroxybutyrate, adding the mixture into the carrier after pore making, and uniformly mixing to obtain a mixture; and (3) performing circulating freeze-thawing and shaping on the mixture, and then putting the freeze-thawed and shaped carrier into a calcium chloride saturated boric acid solution for chemical crosslinking surface layer reinforcement to obtain the immobilized microorganism carrier. The immobilized microbial carrier prepared by the invention has the characteristics of high stability, high microbial activity and macroporosity in the long-time coking wastewater degradation process.

Description

Preparation method of immobilized microorganism carrier, product and application thereof

Technical Field

The invention relates to the technical field of coking wastewater treatment, in particular to a preparation method of an immobilized microorganism carrier, and a product and application thereof.

Background

By the end of 2020, the annual coke yield of China reaches 4.71 million tons, which accounts for about 70% of the global total coke yield, and is the first major coke production, consumption and export country in the world. Based on the theoretical basis that about 0.6 ton of wastewater is generated by one ton of coke, at least 2.83 hundred million tons of coking wastewater are generated in China one year, and the typical refractory organic industrial wastewater has high pollutant load and obvious toxic effect. In a complex coking wastewater environment, the colonization and maintenance of functional degrading bacteria are the basis for realizing the effective biological enhanced degradation of coking wastewater, and if a good biological enhanced effect is to be achieved, the added microorganisms must keep a certain metabolic activity in a wastewater biological treatment system and maintain a certain amount. In the traditional activated sludge biological treatment system, in order to keep the continuity of biological enhancement, thalli are periodically added into a part of wastewater treatment plants to be used as a biological supplement agent, so that the difficulty and the cost of biological enhancement application are inevitably increased. The immobilization technology provides an important means for the efficient bacteria to fully exert the biological potential in the treatment of the organic pollutants difficult to degrade. At present, researchers commonly adopt a method of combining biological reinforcement with an immobilized carrier to carry out the treatment research of the refractory wastewater, and on one hand, the method can effectively solve the defect that free bacteria are directly added and are easy to run off, and improve the stability and effectiveness of biological reinforcement; on the other hand, the biological toxic action of toxic waste water on microorganisms can be relieved. Meanwhile, the immobilized microorganisms are easy to separate solid from liquid, the biological density is high, the degradation efficiency is superior to that of the traditional suspended activated sludge, compared with a biofilm method, the method is easier to control the type and the quantity of the immobilized microorganisms, and the prepared immobilized microorganism carrier can be stored for a long time and is convenient to transport. The embedding immobilization method taking polyvinyl alcohol (PVA) as a base material is an immobilization method which is researched more and has better performance at present, the synthesis theory and the application research thereof are still a leading-edge field, and the method has good development prospect and huge application market. So far, the application research of immobilized microorganism in water treatment for biological enhancement is still in the primary stage, the design and preparation of novel carriers are the key points of the research and development of immobilization technology, and the search for an immobilization method capable of simultaneously meeting the requirements of the stability, mass transfer and microbial activity of the immobilized microorganism carrier is an important direction of the development of immobilization technology.

Disclosure of Invention

The invention aims to provide a preparation method of an immobilized microorganism carrier, a product and an application thereof, aiming at solving the problems in the prior art.

In order to achieve the purpose, the invention provides the following scheme:

the invention provides a preparation method of an immobilized microorganism carrier, which comprises the following steps:

(1) mixing polyvinyl alcohol, sodium alginate and CaCO₃Adding the PVA into deionized water, and heating and dissolving the mixture to be gelatinous to obtain a PVA mixed solution;

(2) adding hydrochloric acid into the PVA mixed solution, and reacting to obtain a carrier after hole making;

(3) mixing the high-efficiency bacterium Comamonas sp.ZF-3 fermentation thallus, activated sludge, powdered activated carbon and polyhydroxybutyrate, adding the mixture into the carrier after pore making, and uniformly mixing to obtain a mixture;

(4) performing circulating freeze-thawing and shaping on the mixture to obtain a freeze-thawed and shaped carrier;

(5) putting the freeze-thawed and shaped carrier into CaCl₂And carrying out chemical crosslinking surface layer reinforcement in a saturated boric acid solution to obtain the immobilized microorganism carrier.

Further, in the step (1), the polyvinyl alcohol, the sodium alginate and the CaCO₃And the mass volume ratio of the deionized water is10 g: 2 g: 1 g: 90 mL.

Further, in the step (1), the heating dissolution is heating dissolution at 90 ℃ for 20 min.

Further, in the step (3), the preparation method of the zymophyte of the high efficiency bacterium Comamonas sp.ZF-3 comprises the following steps: inoculating the high-efficiency bacterium Commamonas sp.ZF-3 into an LB culture medium, and culturing for 16h under the conditions of pH 7.0, 30 ℃ and 130r/min to obtain a fermentation broth of the high-efficiency bacterium Commamonas sp.ZF-3; centrifuging the fermentation liquor, and removing the supernatant to obtain a precipitate; and washing the precipitate by phosphate buffer solution, centrifuging again, and taking the precipitate to obtain the high-efficiency bacterium Comamonas sp.ZF-3 fermentation thallus.

Further, in the step (3), the mass ratio of the zymophyte of the high efficiency bacterium Comamonas sp.ZF-3 to the activated sludge is 1: 4.

further, in the step (3), the bioaugmentation sludge composed of the zymophyte of the high efficiency bacteria Comamonas sp.ZF-3 and the activated sludge is added into the immobilized microorganism carrier in an amount of 28 mg/mL.

Further, in the step (4), the freeze-thaw shaping is cyclic freeze-thaw for 4 times, and each freeze-thaw specifically comprises: the mixture was frozen at-8 ℃ for 20h and then thawed at room temperature for 4 h.

Further, in the step (5), the time for the chemical crosslinking is 70 min.

The invention also provides the immobilized microorganism carrier prepared by the preparation method.

The invention also provides application of the immobilized microorganism carrier in coking wastewater treatment.

The invention discloses the following technical effects:

the immobilized microorganism carrier prepared by the coupling method of circulating freeze thawing, surface boric acid crosslinking and calcium carbonate pore making shows the characteristics of high stability, high microbial activity and macroporosity in the long-time coking wastewater degradation process. The carrier adopts a circulating freeze thawing and short-time surface layer boric acid crosslinking immobilization mode, the inner layer is a space network structure formed by hydrogen bonds between chain molecules and in molecules, the surface is an insoluble gel polymer formed by crosslinking reaction with boric acid, and the carrier has good stability and can still maintain a stable physical form after long-time oscillation operation; meanwhile, the amount of the bacterial sludge prepared by the carrier, the crosslinking time and the freezing and thawing frequency are optimized through modeling by a response surface method, the activity of microorganisms in the carrier is ensured to the maximum extent on the premise that the physical stability and the mechanical strength of the carrier are realized by adopting surface layer crosslinking, the microbial community structure in the carrier is basically kept stable in the whole reaction process, and the purpose of effectively proliferating functional microorganisms including nitrobacteria is realized under the condition that the superior state is continuously kept by high-efficiency bacteria, so that the tolerance and the impact resistance of the immobilized carrier microorganisms to the pollution load impact of the coking wastewater are reflected; compared with a small-pore carrier, the activity space of microorganisms in the carrier is improved by making pores through calcium carbonate, the proliferation and the updating of the microorganisms in the carrier are facilitated, and the problems of carrier pore blockage, poor mass transfer effect of organic pollutants and the like in the microorganism replacement process in the PVA carrier prepared by a boric acid method are solved. The invention provides an effective immobilization scheme for the biological enhanced treatment of the coking wastewater.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings without creative efforts.

FIG. 1 is an internal microbial morphological feature of an immobilization carrier prepared in example 1;

FIG. 2 is a tree of relative abundance taxonomies of major bacterial genera in immobilized microbial carriers;

FIG. 3 shows the effect of the amount of bacterial sludge, cross-linking time, and number of freeze-thaw cycles on the stability of the carrier: (a) the influence of the interaction of the amount of bacterial sludge and the crosslinking time on the stability of the carrier; (b) influence of interaction of the bacterial sludge amount and the freeze-thaw times on the stability of the carrier; (c) the influence of the interaction of the crosslinking time and the freezing and thawing times on the stability of the carrier;

FIG. 4 is a graph showing the effect of the amount of bacterial sludge, cross-linking time, and number of freeze-thaw cycles on the microbial activity of the carrier: (a) the influence of the interaction of the amount of bacterial sludge and the crosslinking time on the microbial activity of the carrier; (b) the influence of the interaction of the bacterial sludge amount and the freeze-thaw times on the microbial activity of the carrier; (c) the influence of the interaction of the crosslinking time and the freezing and thawing times on the microbial activity of the carrier;

FIG. 5 shows the effect of the amount of bacterial sludge, cross-linking time, and freezing and thawing frequency on the comprehensive performance of the carrier: (a) the influence of the interaction of the amount of the bacterial sludge and the crosslinking time on the basic performance of the carrier; (b) the influence of the interaction of the bacterial sludge amount and the freeze-thaw times on the basic performance of the carrier; (c) the influence of the interaction of the crosslinking time and the freezing and thawing times on the basic performance of the carrier;

FIG. 6 shows the synthesis mechanism and chemical characteristics of the immobilized carrier: (a) FT-IR analysis of the carrier synthesis process, wherein heating is the carrier prepared in comparative example 1, heating + freeze-thaw is the carrier prepared in comparative example 2, heating + freeze-thaw + crosslinking is the carrier prepared in comparative example 3, heating + freeze-thaw + crosslinking + pore-making is the carrier prepared in example 1; (b) the chemical structure of the carrier synthesis process is changed;

FIG. 7 shows the apparent morphology change during the synthesis of the immobilized carrier: (a) the support prepared in comparative example 1, (b) the support prepared in comparative example 2, (c) the support prepared in comparative example 3, (d) the support prepared in example 1;

FIG. 8 shows the pore size distribution change during the carrier synthesis: (a) the support prepared in comparative example 1, (b) the support prepared in comparative example 2, (c) the support prepared in comparative example 3, (d) the support prepared in example 1;

FIG. 9 shows COD degrading performance of immobilized microorganism carrier;

FIG. 10 shows NH degradation by immobilized microbial carriers₄ ⁺-N properties;

FIG. 11 is a GC-MS analysis of the effect of the immobilized microorganism carrier on the degradation of organic components: (a) water is fed; (b) degrading bacterial sludge into water; (c) degrading the carrier to obtain water;

FIG. 12 is a comparison of the morphology and internal characteristics of immobilized microbial carriers before and after the degradation reaction of coking water: (a) observing the apparent appearance of the carrier before reaction; (b) observing the apparent appearance of the carrier after reaction; (c) carrying out laser confocal 3D observation on the internal structure of the carrier before reaction; (d) carrying out laser confocal 3D observation on the internal structure of the carrier after reaction; (e) observing the internal appearance of the carrier before reaction; (f) observing the internal appearance of the carrier after reaction;

FIG. 13 shows pore size distribution of immobilized microbial carriers after coking water degradation reaction;

FIG. 14 is a relative abundance classification tree of major bacterial genera in the immobilized microbial carrier after the coking water degradation reaction (the size of the circle represents the abundance of each phylum, class, order and family, and the size of the stars represents the proportion of each genus);

FIG. 15 is a relative abundance classification tree of major bacterial species in bacterial sludge after a coking water degradation reaction;

fig. 16 is a functional difference analysis of the microbial community of the immobilized carrier and the bacterial sludge after the degradation reaction of the coking water (the abundance ratio of different functional abundances in two samples is shown on the left, the difference ratio of the functional abundances is shown in the middle within a 95% confidence interval, the rightmost value is a p value, the p value is less than 0.05, the difference is significant, and the functional abundance is marked by red).

Detailed Description

Reference will now be made in detail to various exemplary embodiments of the invention, the detailed description should not be construed as limiting the invention but as a more detailed description of certain aspects, features and embodiments of the invention.

It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. Further, for numerical ranges in this disclosure, it is understood that each intervening value, between the upper and lower limit of that range, is also specifically disclosed. Every intervening value, to the extent any stated value or intervening value in a stated range, and any other stated or intervening value in a stated range, is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference herein for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control.

It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the present disclosure without departing from the scope or spirit of the disclosure. Other embodiments will be apparent to those skilled in the art from consideration of the specification. The description and examples are intended to be illustrative only.

As used herein, the terms "comprising," "including," "having," "containing," and the like are open-ended terms that mean including, but not limited to.

ZF-3 in the following examples or comparative examples refers to the paper "Treatment of linkage water in biological-based bioavailability process"; the activated sludge (suspended solid concentration MLSS is 3000mg/L) is taken from a secondary sedimentation tank of the wastewater treatment process of a coking plant, is activated for 12 hours in a shaking table at 30 ℃ and is centrifuged for 8min at the rotating speed of 6000r/min, and the supernatant is removed for use. The coking wastewater used in the following effect verification experiment is taken from a coking plant wastewater treatment process regulating reservoir, and the wastewater is pretreated by ammonia distillation, oil removal, air flotation and the like to obtain COD (chemical oxygen demand) and NH (NH) in water₄ ⁺The concentration of-N was 2490mg/L and 189mg/L, respectively.

The preparation method of the high-efficiency bacterium Comamonas sp.ZF-3 fermentation thallus comprises the following steps: inoculating the high-efficiency bacterium Comamonas sp.ZF-3 into an LB culture medium, culturing for 16h under the conditions of pH 7.0, 30 ℃ and 130r/min to obtain a fermentation liquid of the high-efficiency bacterium Comamonas sp.ZF-3, centrifuging the fermentation liquid for 8min under the condition of the rotating speed of 6000r/min, removing supernatant, washing by a phosphate buffer solution (pH 7.4), and centrifuging again to obtain the fermentation thalli of the high-efficiency bacterium Comamonas sp.ZF-3.

Example 1

(1) 10g of 1799 PVA, 2g of Sodium Alginate (SA) and 1g of CaCO₃Adding 90mL of deionized waterHeating the beaker in water bath at 90 deg.C for 20min until the mixture is completely dissolved and becomes gel-like to obtain PVA mixed solution, taking out the beaker from the water bath device, standing and cooling.

(2) When the temperature of the PVA mixed solution is reduced to 50 ℃, 5mL of dilute hydrochloric acid with solute mass fraction of 10% is added into the beaker, and the mixture is rapidly stirred until no air bubbles are generated so that CaCO is generated₃And (3) fully reacting with hydrochloric acid to increase the porosity and the pore uniformity of the carrier, and continuously cooling to 35 ℃ to obtain the carrier after pore making.

(3) Mixing the high-efficiency bacterium Comamonas sp.ZF-3 fermentation thallus with activated sludge according to the mass ratio of 1:4, taking 2.8g as bio-enhanced sludge, uniformly mixing with 1g of Powdered Activated Carbon (PAC) and 1g of Polyhydroxybutyrate (PHB) in advance, adsorbing for 30 minutes, adding into the carrier after hole making, which is cooled to 35 ℃ in the step (2), and fully stirring to obtain a mixture.

(4) Slowly pouring the mixture obtained in the step (3) into a porous silica gel mold with the thickness of 160mm x 160mm at a constant speed, and ensuring that the mixture fills the square hole of the mold without leaving a gap. And (3) covering the mold box filled with the mixture, putting the mold box into a refrigerator at-8 ℃ for freezing for 20h, taking out the mold box and unfreezing for 4h at room temperature (25 ℃), and circularly freezing and thawing for 4 times to obtain the freeze-thaw shaped carrier.

(5) Putting the freeze-thawed and shaped carrier into 2 percent CaCl₂Chemically crosslinking in saturated boric acid solution for 70min, finally washing the newly prepared carrier with deionized water for 3 times, and removing residual boric acid solution to obtain the immobilized microorganism carrier.

As shown in fig. 1, Scanning Electron Microscope (SEM) observation is performed on the internal morphology of the immobilized microorganism carrier prepared in example 1 under different magnifications, and the image shows that the prepared immobilized microorganism carrier has developed internal voids and uniform pore size distribution, and the immobilized microorganisms are mostly embedded in the immobilized carrier pore channels in the form of a cenobium. Meanwhile, the uniformly distributed macropores provide favorable conditions for mass transfer of dissolved oxygen in the immobilized carrier.

Taxonomic tree analysis of the microbial community structure of the immobilized carrier prepared in example 1 was performed by high throughput 16SrRNA gene sequencing technology, as shown in fig. 2, Proteobacteria (85.29%) is the most abundant one in the system, and it includes the following advantages: comamonas (32.61%), Pseudomonas (4.32%), Thauera (2.60%) and Thiobacillus (2.31%), etc. The efficient strain Comamonas sp.ZF-3 accounts for 32.61% in the carrier microbial community, and the successful immobilization of the efficient strain is shown to become the dominant bacterium in the microbial carrier. Pseudomonas, Thauera and Thiobacillus contained in the carrier are common genera of a water treatment system and widely exist in coking wastewater treatment. In addition, Firmicutes (1.65%), Bacteroidetes (5.29%) and Planctomycetes (1.84%) are also present in a certain proportion, these microorganisms being ubiquitous in wastewater treatment and playing a corresponding role. In the nitrifying bacteria aspect, a small proportion of Nitrospira (0.72%) is detected in the immobilized microorganism carrier, and the Nitrospira are responsible for the oxidation of nitrite and play a key role in the denitrification process of wastewater, but because the Nitrospira belongs to autotrophic bacteria, the growth period is long, the requirement on the environment is high, the proportion in activated sludge is reduced, and the embedding proportion is low.

Experimental example 1

The invention adopts a response surface method and a Box-Behnken (BBD) experimental design method to research the influence of the preparation parameters of the immobilized carrier on the carrier performance and calculate the optimal preparation parameters.

Analyzing the chemical structure characteristics of the carrier synthesis process by adopting Fourier transform infrared absorption spectrum (FT-IR), performing functional group and chemical structure analysis by using a Fourier infrared spectrometer Nicolet iS10 FT-IR spectrometer after the carrier iS sliced and freeze-dried, wherein the spectrum scanning range iS 400-4000cm^-1Resolution of spectrometer 4cm^-1The signal-to-noise ratio is 50000:1, and the scanning is carried out 32 times.

The specific surface area of the carrier is tested by a specific surface area test method (BET), the carrier is sliced, frozen and dried, and then a liquid nitrogen adsorption and desorption experiment is carried out by an APAS2460 type full-automatic physical adsorption instrument, and the specific surface area of the carrier is analyzed and measured.

The carrier pore size distribution, porosity, apparent density and other physical characteristics are tested by adopting a mercury intrusion Method (MIP), the carrier is sliced and freeze-dried and then is analyzed by using an AutoPore lv9510 high-performance full-automatic mercury intrusion instrument, the test temperature is room temperature, the maximum pressure of high-pressure analysis is 60000psia, the corresponding minimum detectable pore size is 3nm, the pressure range of low-pressure analysis is 0.5-50psia, the corresponding detectable pore size range is 360-3.6 mu m, and the detected mercury ingress/egress amount is as small as 1 mu L.

Carrying out morphology observation on the carrier sample by adopting a Scanning Electron Microscope (SEM), wherein the carrier sample is required to be subjected to sample preparation treatment before the SEM observation: placing a sample in 2.5% glutaraldehyde solution for 2h at 4 ℃, washing for 3 times by using phosphoric acid buffer solution, dehydrating by using 30%, 50%, 70%, 80% and 90% ethanol for 15min in sequence, dehydrating by using 100% ethanol for 20min, replacing for two times by using isoamyl acetate, freeze-drying for 24h, and spraying gold for observation under 15kv working voltage.

And (3) analyzing the biological community inside the immobilized carrier by adopting a high-throughput 16SrRNA gene sequencing technology. The vector samples were obtained from the inside of the vector using a sterile razor blade, and the samples were subjected to gene extraction amplification after freeze-drying at-50 ℃ (BT2KXL, Virtis, USA) and grinding with sterile ceramic.

The invention adopts a circulating freeze thawing-surface boric acid crosslinking-calcium carbonate hole making coupling method to prepare the immobilized carrier, and the main independent variable factors influencing the carrier performance in the preparation parameters comprise the amount of immobilized bacterial sludge, the crosslinking time and the freeze thawing frequency. Referring to the preparation method of example 1, the amount of the bacterial sludge (unit mg/mL, namely, the content of the biologically enhanced sludge in the mixture obtained in step (3) of example 1, the crosslinking time and the number of freeze-thaw cycles are selected as three independent variables, the stability of the immobilized microorganism carrier and the activity of the microorganism in the immobilized carrier are respectively taken as response variables, an experimental Design is performed by adopting a Response Surface (RSM) three-factor Box-Behnken Design (BBD) method to obtain an optimal regression model, each independent variable factor influencing the stability of the immobilized microorganism carrier and the activity of the microorganism in the immobilized carrier is quantitatively analyzed, the influence of the interaction of the independent variable factors on the stability of the immobilized microorganism carrier and the activity of the microorganism in the carrier is analyzed, and the level and the range of each independent variable are listed in table 1.

TABLE 1 levels and ranges of independent variables and code values in BBD (Box-Behnken) design

Table 2 BBD experiment design scheme and results of influence of preparation parameters on vector stability

17 groups of immobilized microorganism carrier stability exploration experiments are obtained through the Design of a response surface three-factor Box-Behnken Design (BBD) experiment scheme. The stability of the carrier is characterized by the Total Organic Carbon (TOC) loss of the carrier in water for 10 days, and the specific operation is that 17 experimental group carriers are immersed into deionized water, shaking is carried out on a table under the constant temperature condition of 30 ℃, samples are taken after 10 days to analyze the TOC concentration in the water, and therefore the amount of the dissolved carrier is calculated. The lower its dissolved concentration in water indicates the lower the amount of TOC loss of the support, the better the support stability. The BBD experimental design scheme and experimental results are shown in Table 2.

And analyzing the experimental data in the table 2 by using Design-Expert 8.06 software, selecting a Quadratic model for correlation, and performing variance analysis on each coefficient of the model in the correlation process. Analysis of variance indicates that an F value of 15.79 for this model means that the model is significant, and a "Prob > F" value of less than 0.0500 indicates that the model terms are valid, with A, B, C, B2, C2 being important model terms. And drawing a response surface 3D stereo analysis diagram and a contour diagram according to Design-Expert 8.05 software to obtain the influence of three factors of the bacterial sludge amount, the crosslinking time and the freezing and thawing frequency on the response variable carrier stability, as shown in FIG. 3.

As shown in FIG. 3(a), when the number of freeze thawing is fixed, the TOC loss of the carrier in water increases with the increase of the bacterial sludge amount, the stability of the carrier decreases, and the decrease of the stability is particularly obvious when the bacterial sludge amount is more than 25mg/mL, as can be seen from the density of the contour lines on the bacterial sludge amount axis. On the contrary, the extension of the crosslinking time can significantly improve the stability of the carrier, and the improvement is most remarkable within the first 45min, as can be seen from the density of the contour lines on the axis of the crosslinking time; as shown in FIG. 3(b), the improvement of the stability of the carrier by the number of freeze-thawing times is mainly concentrated in the first three times, and the influence of freeze-thawing more than three times on the stability of the carrier is not obvious, which is reflected in the sparsity of contour lines on the axis of the number of freeze-thawing times. Meanwhile, the contour map is observed to be elliptic, which shows that the quantity of the bacterial sludge and the freezing and thawing times have certain interaction effect on the stability of the carrier; FIG. 3(c) shows that cross-linking time and freeze-thaw times also have some interactive effects on carrier stability.

TABLE 3 BBD Experimental design protocol and results for Effect of preparative parameters on Carrier microbial Activity

Through the Design of a response surface three-factor Box-Behnken Design (BBD) experimental scheme, 17 groups of immobilized microorganism activity exploration experiments are obtained. The microbial activity in the immobilized carrier can be represented by a Chemical Oxygen Demand (COD) experiment for degrading coking wastewater by a carrier shake flask, and the specific operation is that 17 experiment group carriers are added into a conical flask containing 150mL of coking wastewater diluted by 3 times, the conical flask is oscillated by a shaker at the constant temperature of 30 ℃, and after 48 hours of degradation at the rotating speed of 130r/min, effluent samples are taken for COD concentration detection, so that the COD content of microbial degradation in the immobilized carrier is judged. The lower the COD of the effluent, the stronger the microbial activity in the immobilized microbial carrier. The BBD experimental design scheme and experimental results are shown in Table 3.

And analyzing the experimental data in the table 3 by using Design-Expert 8.06 software, selecting a Quadratic model for correlation, and performing variance analysis on each coefficient of the model in the correlation process. Analysis of variance showed that an F value of 1593.9 for this model means that the model is significant, "Prob>An F "value less than 0.0500 indicates that the model term is valid, where A, B, AB, A²、C²Are important model terms. Drawing response surface 3D stereo analysis according to Design-Expert 8.06 softwareAnd obtaining the influence of three factors of the bacterial sludge amount, the cross-linking time and the freezing and thawing frequency on the microbial activity in the response variable immobilized carrier by the diagram and the contour diagram.

As shown in FIG. 4(a), when the number of times of freezing and thawing is fixed, the COD in the effluent from the degradation experiment decreases with the increase of the bacterial sludge amount, which indicates that the activity of the microorganisms in the immobilized carrier is higher, and when the bacterial sludge amount is greater than 25mg/mL, the COD in the effluent decreases relatively slowly. On the contrary, the activity of microorganisms in the carrier can be reduced by prolonging the crosslinking time, the influence is obvious after 63min, and the contour diagram is elliptic as can be seen from the density of contour lines on the axis of the crosslinking time, which shows that the microbial sludge amount and the crosslinking time have certain interaction influence on the activity of the microorganisms in the immobilized carrier; as shown in fig. 4(b), the freezing and thawing times have a negligible effect on the microbial activity in the vector; FIG. 4(c) shows that cross-linking time and freeze-thaw times have some interactive effect on microbial activity in the vehicle, but the freeze-thaw times have no significant effect on microbial activity in the vehicle.

Table 4 BBD experiment design scheme and results of influence of preparation parameters on comprehensive properties of carrier

The research shows that the embedded bacteria mud amount, the cross-linking time and the circulating freeze-thaw frequency are taken as three independent variable factors, and direct influence or interactive influence is generated on the stability of the immobilized microorganism carrier and the activity of microorganisms in the immobilized carrier. The preparation of the immobilized microorganism carrier needs to consider both the stability of the carrier and the activity of microorganisms in the carrier, on the basis, the response variable of the comprehensive performance of the carrier is a comprehensive combination of the stability of the carrier and the activity of the microorganisms in the carrier, according to the numerical difference between the TOC loss and the COD effluent, 4TOC + COD is taken as the representation of the comprehensive performance of the immobilized carrier, the smaller the numerical value of 4TOC + COD is, the lower the TOC loss and the COD effluent are, the better the stability of the carrier and the activity of the microorganisms in the carrier are, and the better the comprehensive performance of the carrier is. Selecting the amount of the bacterial sludge, the cross-linking time and the freezing and thawing times as three independent variables, taking the comprehensive performance of the immobilized microorganism carrier as a response variable to obtain an optimal regression model, seeking an optimal condition by using the model, analyzing the influence of the interaction of the independent variable factors on the comprehensive performance of the immobilized microorganism carrier, and processing the experimental data to obtain a BBD experimental design scheme and experimental results shown in Table 4.

And analyzing the experimental data in the table 4 by using Design-Expert 8.06 software, associating by using a Quadratic model, and carrying out variance analysis on each coefficient of the model in the association process. Analysis of variance showed that an F value of 42.43 for this model means that the model is significant, "Prob>An F "value less than 0.0500 indicates that the model term is valid, where A, B, AB, A²、B²、C²Are important model terms. The coefficient of distortion significance, Lack offset, is 3.21(Lack offset)>0.05) not significant. And analyzing and drawing a response surface 3D analysis chart and a contour map according to Design-Expert 8.05 software to obtain the influence of three factors of the bacterial sludge amount, the crosslinking time and the freezing and thawing times on the comprehensive performance of the response value immobilized carrier.

As shown in FIG. 5(a), when the number of times of freezing and thawing is fixed, 4TOC + COD decreases first and then increases with the increase of the amount of bacterial sludge, and the corresponding comprehensive performance of the carrier increases first and then decreases, which indicates that the amount of bacterial sludge is mainly shown as the improvement of the degradation activity of the microorganism in the immobilized carrier before about 30mg/mL, and the adverse effect of the amount of bacterial sludge on the stability of the immobilized microorganism carrier after 30 mg/mL. Similarly, along with the increase of the cross-linking time, the 4TOC + COD is firstly reduced and then increased, and the corresponding comprehensive performance of the carrier is firstly increased and then reduced, which shows that the cross-linking time of the bacteria is mainly shown as the improvement of the stability of the immobilized carrier before 72min, the cross-linking time of the bacteria is mainly shown as the influence on the activity of the immobilized microorganism carrier after 72min, and the contour diagram is an ellipse, which shows that the bacterial sludge amount and the cross-linking time have certain interaction influence on the comprehensive performance of the immobilized carrier; as shown in fig. 5(b), with the increase of the number of times of freezing and thawing, 4TOC + COD decreased first and then increased, and the corresponding comprehensive performance of the carrier increased first and then decreased, which indicates that the number of times of freezing and thawing before 4 times is mainly shown as improvement of the stability of the immobilized carrier, and after 4 times is mainly shown as influence on the activity of the immobilized microorganism carrier, and the contour diagram is an ellipse, which indicates that the amount of the bacterial sludge and the number of times of freezing and thawing have a certain interaction effect on the comprehensive performance of the immobilized carrier; FIG. 5(c) shows that cross-linking time and freeze-thaw times have some, but not significant, interactive effects on the overall performance of the carrier.

After the regression fitting, the relationship between each factor and the response value can be expressed by the following regression equations (1-1) and (1-2).

Equation derived from the coding factor:

4TOC+COD＝446.06-107.84A-82.90B-51.76C+64.33AB+27.80AC-16.22BC+222.80A²+72.47B²+65.34C² (1-1)

an equation derived from practical factors:

4TOC + COD (1238.42000-26.37810) bacterial sludge amount-5.95172 crosslinking time-129.68625 times of freezing and thawing +0.057178 bacterial sludge amount-crosslinking time +0.55600 times of bacterial sludge amount-0.18028 times of crosslinking time +0.35647 times of freezing and thawing²+0.035788 Cross-linking time²+16.33625 freeze thaw cycles² (1-2)

The equation correlation coefficient R is 0.7883, which shows that the fitting equation has high accuracy and small experimental mismatching terms and can be used for analyzing actual values. Solving the optimal preparation parameters of the immobilized microorganism carrier by the regression equation, namely solving the value of the independent variable when the minimum value of 4TOC + COD is solved, and solving the optimal conditions as follows: the bacterial sludge amount is 28mg/mL, the cross-linking time is 70min, and the freezing and thawing times are 4 times.

Comparative example 1

(1) 10g of 1799 PVA, 2g of Sodium Alginate (SA) and 1g of CaCO₃Adding into a beaker containing 90mL of deionized water, heating at 90 deg.C for 20min under stirring in water bath until the mixture is completely dissolved and becomes gel-like to obtain PVA mixed solution, taking out the beaker from the water bath device, standing and cooling.

(2) Mixing the high-efficiency bacterium Comamonas sp.ZF-3 fermentation thallus with activated sludge according to the mass ratio of 1:4, taking 2.8g as bio-enhanced sludge, uniformly mixing with 1g of Powdered Activated Carbon (PAC) and 1g of Polyhydroxybutyrate (PHB) in advance, adsorbing for 30 minutes, adding into the PVA mixed solution cooled to 35 ℃ in the step (1), and fully stirring to obtain the immobilized microorganism carrier.

Comparative example 2

(2) Mixing the high-efficiency bacterium Comamonas sp.ZF-3 fermentation thallus with activated sludge according to the mass ratio of 1:4, taking 2.8g as bio-enhanced sludge, uniformly mixing with 1g of Powdered Activated Carbon (PAC) and 1g of Polyhydroxybutyrate (PHB) in advance, adsorbing for 30 minutes, adding into the PVA mixed solution cooled to 35 ℃ in the step (1), and fully stirring to obtain a mixture.

(3) Slowly pouring the mixture obtained in the step (2) into a porous silica gel mold with the thickness of 160mm x 160mm at a constant speed, and ensuring that the mixture fills the square hole of the mold without leaving a gap. And (3) covering the mold box filled with the mixture, putting the mold box into a refrigerator with the temperature of-8 ℃ for freezing for 20h, taking out the mold box, thawing for 4h at room temperature, and circularly freezing and thawing for 4 times to obtain the immobilized microorganism carrier.

Comparative example 3

(3) Slowly pouring the mixture obtained in the step (2) into a porous silica gel mold with the thickness of 160mm x 160mm at a constant speed, and ensuring that the mixture fills the square hole of the mold without leaving a gap. And (3) covering the mold box filled with the mixture, putting the mold box into a refrigerator at-8 ℃ for freezing for 20h, taking out the mold box, thawing for 4h at room temperature, and performing freeze thawing for 4 times in a circulating manner.

(4) Putting the freeze-thawed and shaped carrier into 2 percent CaCl₂Chemically crosslinking in saturated boric acid solution for 70min, finally washing the newly prepared carrier with deionized water for 3 times, and removing residual boric acid solution to obtain the immobilized microorganism carrier.

The immobilized microorganism carriers prepared in comparative examples 1-3 and example 1 were characterized by Fourier transform infrared absorption spectrometer (FTIR) to explore the chemical structure characteristics during the carrier synthesis process and analyze the synthetic chemical basis.

As shown in FIG. 6(b), the polyvinyl alcohol (PVA) has the chemical structure of polyol, and can be regarded as polyol containing hydroxyl active groups on alternate carbon atoms, the completely alcoholyzed PVA (alcoholysis degree is more than or equal to 98%) can be completely dissolved only by heating to 80 ℃ in water, and the completely dissolved homogeneous colloidal solution is obtained by heating the water solution in a 90 ℃ water bath, which shows that the PVA has good hydrophilicity under heating conditions.

As shown in FIG. 6(a), the FT-IR spectrum of the sample after the heat treatment was 3250cm^-1、2905cm^-1、1601cm^-1、1415cm^-1、1321cm^-1、1081cm^-1Six absorption peaks appear and are attributed to hydroxyl (-OH) and methylene (-CH) in PVA₂-), C-O, etc., indicating that the chemical structure of the PVA solution remains stable after heat treatment.

The hydroxyl groups have small size and strong polarity, and are easy to form hydrogen bonds, the hydroxyl groups are modified by a freeze-thaw method, and the FT-IR spectrum of a sample subjected to heating and cyclic freeze-thaw treatment is 3150cm as shown in FIG. 6(a)^-1The absorption peak attributed to hydrogen bond (O.H) appears, which shows that the PVA solution forms three-dimensional networks of hydrogen bond and microcrystal region among and in chain molecules after heating and circulating freeze thawing, and the chemical structure is shown in figure 6 (b).

The PVA is modified by adopting a boric acid crosslinking method, and the FT-IR spectrum of a sample after heating, circulating freeze thawing and boric acid crosslinking is 1283cm in figure 6(a)^-1The occurrence of a bond belonging to the B-O bondThe peak is shown, the covalent bond of B-O is generated by the reaction of hydroxyl in the PVA solution and boric acid ion, the reaction equation is shown in figure 6(B), and the reaction time is 3250cm^-1The absorption peak indicates that hydroxyl (-OH) groups still exist and the crosslinking reaction does not stay completely in the surface structure.

The principle of calcium carbonate pore-forming is that dilute hydrochloric acid reacts with calcium carbonate pore-forming agent in a carrier to release carbon dioxide in the stirring process, the internal air pressure of carbon dioxide bubbles and the pressure of PVA solution reach balance, the bubbles are difficult to overflow due to diffusion resistance, and stable and uniform holes are formed in the carrier to improve the carrier mass transfer performance, as shown in fig. 6(a), the FT-IR spectrum of a sample is not obviously changed after pore-forming, which indicates that pore-forming has no influence on the chemical structure of the carrier.

In conclusion, under the optimal preparation parameters, the immobilized microorganism carrier prepared by the method of circulating freeze thawing-surface boric acid crosslinking-calcium carbonate pore-making coupling is adopted, the inner layer is a space network structure formed by hydrogen bonds between chain molecules and in molecules, the surface is an insoluble gel polymer formed by the crosslinking reaction with boric acid, the immobilized microorganism carrier has good stability, and meanwhile, the chemical structure of the carrier is not influenced by pore-making.

The immobilized microorganism carriers prepared in comparative examples 1-3 and example 1 were characterized by Scanning Electron Microscope (SEM), specific surface area tester (BET), mercury porosimetry pore test to investigate the physical property characteristics during the synthesis thereof, and the results are shown in fig. 7, fig. 8, and table 5.

As shown in FIG. 7(a), the PVA solution has good film-forming properties after heating, and apparently no obvious pore channel appears, and FIG. 8(a) also shows that the sample has no obvious pore size distribution after heating. Table 5 shows that the BET specific surface area of this sample is 1.0740m²Per g, Porosity of 11.7031%, apparent Density of Applent (skelestal) Density of 1.1.3584 g/mL.

As shown in FIG. 7(b), the sample after cyclic freeze-thawing apparently shows a porous net-shaped structure due to the intermolecular and intramolecular hydrogen bonds of chains and the three-dimensional network of microcrystalline regions formed by cyclic freeze-thawing, FIG. 8(b) shows that the sample after cyclic freeze-thawing has a pore size distribution in the range of 100-1000 nm, and Table 5 shows that the BET specific surface area of the sample is rapidly enlarged by about ten times to 10.7358m²(iv)/g, Median Pore Diameter (V) is 325.9nm, Porosity increases to 41.1221%, and apparent Density application (skelestal) Density decreases to 1.1622 g/mL.

The hydroxyl in the sample after chemical crosslinking reacts with boric acid ions to generate B-O covalent bonds in a single-glycol type, the apparent structure of the sample is more compact in a porous network structure and can be attributed to the porous structure formed by covalent bonding, the pore diameter of the sample after cyclic freeze thawing is shifted to 10-100 nm as shown in fig. 7(c), and the increase of the BET specific surface area of the sample is not obvious 11.7276m in table 5²(iv)/g, Median Pore Diameter (V) of 130.5nm, Porosity reduction to 38.0594%, apparent Density application (skelestal) Density of 1.0455 g/mL.

As shown in FIG. 7(d), the porous network structure of the calcium carbonate-made pore sample after freeze-thaw crosslinking is distributed with pores with larger pore diameters, FIG. 8(d) shows that the sample has a large pore diameter distribution of 1000-10000 nm, and Table 5 shows that the BET specific surface area of the sample also rapidly increases to 15.8477m²The Median Pore Diameter (V) is 2266.2nm, the Porosity increases to 64.6818%, and the apparent Density applied (skelestal) Density decreases to 0.9932g/mL, which is lower than the water Density.

In conclusion, under the optimal preparation parameters, the immobilized microorganism carrier prepared by adopting the circulating freeze-thawing-surface boric acid crosslinking-calcium carbonate pore-making coupling method has the advantages that the inner layer is of a space network structure formed by hydrogen bonds between chain molecules and in molecules, the surface is an insoluble gel polymer formed by crosslinking reaction with boric acid, the immobilized microorganism carrier has good stability, a large amount of pore size distribution of 1000-10000 nm appears in the carrier after pore making by calcium carbonate, and the BET specific surface area is also rapidly increased to 15.8477m²And g, the Median Pore Diameter (V) is 2266.2nm, the Porosity is improved to 64.6818%, the apparent Density (apparent) Density is reduced to 0.9932g/mL, and the carrier is successfully prepared and has a good macroporous structure.

TABLE 5 physical Properties of the immobilized support Synthesis Process

Effect verification

1. Analytical method

(1) Degradation effect analysis method

In the immobilized carrier precipitation experiment, ammonia Nitrogen (NH) in inlet and outlet water₄ ⁺-N) and Chemical Oxygen Demand (COD) concentrations were determined using the Nagowski reagent spectrophotometry and dichromate method, and organic contaminants in water were determined using solid phase microextraction-gas chromatography mass spectrometry (SPME-GC-MS).

(2) Method for analyzing characteristics of immobilized carrier

The internal structure of the carrier was observed using a Leica TCS-SP8 Confocal Laser Scanning Microscope (CLSM) with excitation wavelengths set at 480 and 520 nm. Before observation under CLSM, the immobilized carrier samples were stained in a dark room at 25 ℃ for 20min with a Live/Dead back Bacterial Viability Kit L7012(Invitrogen, USA) Kit containing two fluorescent nucleic acid markers SYTO9 and Propidium Iodide (PI), which were stained to penetrate intact cells (stained with green fluorescence) and damaged cells (stained with red fluorescence), respectively; observing the apparent morphology of the carrier by using a Scanning Electron Microscope (SEM), wherein before the SEM observation, a sample preparation treatment needs to be carried out on an immobilized carrier sample: placing a sample in 2.5% glutaraldehyde solution for 2h at 4 ℃, washing for 3 times by using phosphoric acid buffer solution, dehydrating by using 30%, 50%, 70%, 80% and 90% ethanol for 15min in sequence, dehydrating by using 100% ethanol for 20min, replacing for two times by using isoamyl acetate, freeze-drying for 24h, and spraying gold for observation under 15kv working voltage. The physical characteristics of the carrier such as specific surface area, pore size distribution, porosity, apparent density and the like are tested by a mercury intrusion Method (MIP) and a BET method.

(3) Immobilized carrier microbial community analysis method

And (3) analyzing the biological community inside the immobilized carrier by adopting a high-throughput 16SrRNA gene sequencing technology. Prior to analysis, the mixed immobilized carrier samples were freeze-dried at-50 deg.C (BT2KXL, Virtis, USA) and ground with sterile ceramic, and the carrier sample genome DN was extracted using the E.Z.N.A.soil.DNA kit (OMEGA Bio-Tek, USA)A and DNA integrity was checked with agarose gel. The vector sample 16SrRNA gene was PCR amplified using V3-V4 region with universal primers 341F (5 '-CCTAGGGNGGCWGCAG-3') and 805R (5 '-GACTACHVGGGTATCTAATCC-3'): pre-denaturation at 94 ℃ for 3min, denaturation at 94 ℃ for 30s, annealing at 45 ℃ for 20s, and extension at 65 ℃ for 30s, after the above steps are cycled for 3 times, denaturation at 94 ℃ for 20s, annealing at 55 ℃ for 20s, and extension at 72 ℃ for 30s, after the above steps are cycled for 20 times, final extension at 72 ℃ for 5min, and then introducing Illumina bridge PCR compatible primers for second PCR amplification: pre-denaturation at 95 ℃ for 3min, denaturation at 94 ℃ for 20s, annealing at 55 ℃ for 20s, extension at 72 ℃ for 30s, circulating the steps for 5 times, finally extension at 72 ℃ for 5min, and purifying the PCR product by Agencour AMPure XP treatment. Use of

3.0 quantification by fluorometer (Life Invitrogen) and homogenization of the same volume, the amplification products were sequenced by the Illumina Miseq PE300 System, Shanghai, Japan, Industrial technologies, Inc. (Shanghai, China). The obtained sequences were clustered into Operational Taxonomic Units (OTUs) at a 97% similarity level using the Mothur program (http:// www.Mothur.org), and Alpha diversity analysis including sparse curve, Chao1 abundance estimation index, Shannon diversity index, Simpson index, and Coverage analysis was performed on each sample to measure sample species diversity and Coverage.

(4) Function prediction of immobilized carrier microbial community

The function of the immobilized carrier microbial community is predicted by adopting a PICRUSt tool, and the PICRUSt tool is named as a Phylogenetic invasion of microorganisms by Reconstruction of unknown States. The tool infers the gene function spectrum of the common ancestor of the tested bacteria 16SrDNA based on the information of the tested bacteria 16SrDNA and the OTU information of the closely related species after comparing the Greenene database, and infers the gene function spectrum of other untested species in the Greenene database at the same time to construct the gene function prediction spectrum of the archaea and the bacterial domain whole lineage; and finally, mapping the flora composition obtained by sequencing into a database, and then predicting the metabolic function of the flora. The accuracy of the method is 84% -95%, and the functional gene composition in the sample can be reflected very well. In addition, based on the KEGG metabolic function database and the PICRUSt function secondary classification result, the functional difference analysis of community functions among samples or groups can be carried out, the functional classification with obvious difference of abundance among samples or groups can be found, and the default screening condition is that P is 0.05.

2. Degradation experiments

Putting all the immobilized microorganism carriers prepared in the example 1 into a conical flask containing 500mL of coking wastewater, carrying out a table shaking degradation test at the rotating speed of 130r/min at the temperature of 30 ℃, carrying out the degradation test for 123 days, sampling and detecting every 48 hours, and replacing all the wastewater; meanwhile, 2.8g of bacterial sludge (wherein the mass ratio of the high-efficiency bacteria Comamonas sp.ZF-3 fermentation thalli to the activated sludge is 1:4, which is consistent with the amount of the bacterial sludge used in example 1) is put into another conical flask containing 500mL of coking wastewater, a control group degradation test is carried out under the same conditions, and the performance of the immobilized carrier for treating the coking wastewater is researched.

2.1COD and Ammonia Nitrogen removal Effect

COD and ammonia nitrogen concentration detection is carried out on the sampling of the conical flask for the degradation experiment every 48 hours, and the result is shown in figure 9 and figure 10.

FIG. 9 shows the COD concentration of the influent water and effluent water and the removal rate thereof as a function of time during the test for degrading coking wastewater by immobilized carriers and bacterial sludge. The test was divided into three stages according to the influent water concentration. The first stage (days 1-13) is a carrier acclimation wastewater stage, diluted coking wastewater (COD 312, NH)₄ ⁺24, mg/L) every 48h, and in the 6 water changing treatment processes at intervals of 48h, the immobilized carriers and the bacteria sludge microorganisms are gradually adapted to the quality of inlet water, which is shown in that the COD concentration is rapidly reduced, the removal rate is rapidly increased, and the increase speed of the removal rate of the immobilized carriers COD gradually exceeds the bacteria sludge from behind. From the second stage (13 th to 63 th days), the COD concentration of the influent sewage is increased continuously, the removal rate of the carrier COD can be maintained above 90 percent basically, the removal rate of the bacterial sludge COD fluctuates obviously and declines in 63 days, and the removal rate of the carrier COD fluctuates obviously in 63 days. At the beginning of the third stage (days 63-123), the COD removal rate of the carrier and bacterial sludge is reduced, and the bacterial sludge reduction is obvious, which means that the fast-rising load of the coking wastewater causes the microorganisms in the carrierThe product also has certain inhibiting effect, but the inhibiting effect is weaker than bacterial sludge. After the increase speed of the COD concentration is slowed down, the removal rate of the COD in the carrier gradually rises and recovers, which shows that the microorganism in the immobilized carrier has good impact resistance and adaptability, and the degradation performance of the bacterial sludge is not obviously recovered. After 109 days, the COD and ammonia nitrogen concentration of the inlet water reaches 2490mg/L and 189mg/L, the removal rate of the immobilized carrier COD is basically stabilized at 92%, and the bacterial sludge is about 50%.

FIG. 10 shows the inlet and outlet NH of the test process for degrading coking wastewater by immobilized carrier and bacterial sludge₄ ⁺The variation of the N concentration and its removal rate over time. The first stage (days 1-13) is a carrier and sludge adaptation wastewater stage, diluting the coking wastewater (COD 312, NH)₄ ⁺24, mg/L) every 48h, and in the 6 water-changing treatment processes at intervals of 48h, the immobilized carriers and the bacterial sludge microorganisms do not show obvious ammonia nitrogen removal rate increase and have fluctuation, and bacterial sludge degradation performance is relatively good. Starting from the second stage (days 13-63), water NH is fed in₄ ⁺Increasing concentration of-N, carrier NH₄ ⁺The removal rate of-N is slowly increased, and the bacterial sludge NH₄ ⁺The N removal rate has a decreasing trend of fluctuation. At the beginning of the third phase (days 63-123), the carrier and bacterial sludge NH₄ ⁺the-N removal rate fluctuates, and the quickly increased load of the coking wastewater has an inhibiting effect on nitrifying bacteria in the carrier. In slowing down NH₄ ⁺After increasing the N concentration rate, the carrier NH₄ ⁺The removal rate of-N is basically stable, which indicates that the nitrifying bacteria in the immobilized carrier can colonize and grow on the carrier, the degradation performance of the bacterial sludge is not obviously recovered, and the low removal rate indicates that the nitrifying bacteria in the bacterial sludge are seriously inhibited and the activity is reduced. After 109 days, the COD and ammonia nitrogen concentration of the inlet water reach 2490mg/L and 189mg/L, and the carrier NH₄ ⁺The removal rate of-N is basically stabilized at about 60%, and the bacterial sludge is lower than 10%.

2.2 degradation Effect of organic contaminants

At day 123, water in and out samples were taken and analyzed for organic content by GC-MS, with the results shown in fig. 11 and table 6. As can be seen from fig. 11 and table 6, the main organic components in the coking wastewater include phenolic compounds such as phenol, 2-methylphenol, 3-methylphenol, 2, 3-dimethylphenol, 3, 5-dimethylphenol, heterocyclic compounds such as quinoline, isoquinoline, indole, and polycyclic aromatic hydrocarbons such as 1-methylnaphthalene, 2-methylnaphthalene, 1, 5-dimethylnaphthalene, acenaphthene, anthracene, carbazole, etc., these organic pollutants are substantially removed by degradation with immobilized microbial carriers, only a small amount of degradation products such as 6-undecanone, refractory organic substances such as 1-methylnaphthalene, 1, 5-dimethylnaphthalene, and plastic residue 2, 4-di-tert-butylphenol are detected in the effluent, and the degradation results of the organic substances are well matched with the degradation data of COD removal. And a large amount of phenolic compounds such as phenol, 2-methylphenol, 3-methylphenol and 3, 5-dimethylphenol, heterocyclic compounds such as quinoline and indole, and polycyclic aromatic hydrocarbons such as dibenzofuran, anthracene and carbazole and the like still exist in the bacterial sludge effluent sample, which shows that the bacterial sludge biodegradation organic pollutant performance is weaker than that of carrier microorganisms, and the concentration of the effluent organic pollutant is high.

TABLE 6 GC-MS search results for the effect of degrading organic components by immobilized microbial carriers

Note: ND stands for not detected.

3. Analysis of immobilized Carrier Properties

3.1 Carrier morphology Structure Change

The apparent morphology and internal features of the immobilized carrier before and after the reaction of the coking water degradation experiment were compared (fig. 12). As shown in FIGS. 12(a) and 12(b), after 123 days of shaking flask experiments, the carrier was structurally stable in appearance, and no breakage or swelling occurred, indicating that the carrier had stable mechanical strength. The fluorescent staining 3D observation and the morphology observation are carried out on the inside of the immobilized carrier by using a laser confocal microscope and a scanning electron microscope, the comparison of the graphs in fig. 12(c) and 12 (e) and 12(D) and (f) shows that the microorganisms in the carrier undergo further growth and reproduction in the process of degrading coking wastewater, the number of the microorganisms in the carrier is obviously increased, the microorganisms in the carrier can still keep good activity after the reaction of the carrier with high-load coking wastewater, and simultaneously, the mass transfer effect in the carrier is indirectly shown to be good, and the good circulation of organic matters such as pollutants and the like in the carrier provides basic material conditions for the proliferation of the microorganisms.

3.2 Carrier physical Property Change

The pore size distribution of 100-10000 nm still exists in the carrier after 123 days of reaction as shown in fig. 13, a good macroporous structure can be maintained, but the amount of macropores of the carrier is obviously reduced compared with the amount of macropores of the carrier before the reaction, which is probably caused by the fact that a large amount of microorganisms proliferate and grow into macropores in the degradation experiment process, and table 7 shows that the BET specific surface area of the carrier after 123 days of operation is 13.7420m²(iv)/g, Median Pore Diameter (V) 1804.5nm, Porosity 51.4698%. The parameters are slightly reduced relative to the carrier before the reaction, but the reduction degree is limited, and the physical characteristics of the carrier are basically stable.

TABLE 7 change in physical Properties after reaction of immobilized Carrier

3.3 vector microbial community abundance and diversity

TABLE 8 microbial community abundance and diversity index of immobilized carrier before and after reaction

^aSeqnum-the number of effective sequences obtained in a sample.

^bOTUnum is the number of operation classification units, and the larger the number is, the richer the community diversity is.

^cShannonindex is one of the microbial diversity indices of the samples, with higher indices indicating more abundant community diversity. .

^dChao1 index is the microbial abundance index of the sample, with higher indexes indicating a richer community.

^eCoverage of the sample-constructed sequence library, higher values indicating a lower probability that the sequence in the sample was not detected.

^fSimpson is one of the microbial diversity indices of the samples, with lower indices indicating greater diversity of the community.

And (3) analyzing the immobilized carrier after the reaction and the microbial community in the bacterial sludge by adopting a high-throughput 16SrRNA gene sequencing technology. Meanwhile, a sample which does not participate in degradation is used as a sample before reaction for comparison analysis, and the diversity and abundance change of microbial communities in the carrier and the bacterial sludge before and after the degradation experiment are researched. The sequencing results in Table 8 show that 51905 and 67333 qualified sequences are obtained from the carrier not participating in the degradation experiment and the carrier reacting for 123 days respectively, the coverage rate is over 95 percent, and the constructed sequence library can reflect the diversity of microbial communities. According to effective readings, 4154 OTUs and 5081 OTUs are observed in total, and the Shannon index, the Chao1 index and the Simpson index show that after the degradation experiment is carried out for 123 days, the abundance and the diversity of the microbial communities in the carriers are improved, the 3D observation result of laser confocal inside the carriers is further supported, and the abundance and the diversity of the microbial communities in the bacterial sludge are reduced relative to the unreacted carriers, which shows that the bacterial sludge is inhibited in the reaction process, and the diversity and the abundance of the microbial communities are reduced.

3.4 succession of microbial flora structures on vectors

And (3) performing classification tree analysis on the microbial community structures of the immobilized carriers and the bacterial sludge after reaction. As shown in FIGS. 14 and 15, Proteobacteria (68.69%) was the most abundant phylum in the vector. Proteobacteria include the following advantageous genera: comamonas (23.25%), Pseudomonas (5.09%), Thiobacillus (6.31%), Diaphorobacter (3.23%), Thauera (2.12%) and Tepidiphilus (2.19%). Wherein, the high efficiency bacterium Comamonas sp.ZF-3 accounts for 23.25% of the vector community after the reaction, and still maintains the dominant state, and the reduction of the proportion of the high efficiency bacterium to the vector before the reaction is caused by the increase of the diversity of the vector microorganisms. Sequencing results show that most of dominant bacteria belonging to Proteobacteria in Proteobacteria in the immobilized carrier can be maintained in the carrier and are relatively stable after 123 days of degradation experiments. The stabilization of the abundance of these genera is the reason why the immobilized microorganism carriers achieve effective degradation of the coking wastewater COD. Efficient bacteria Comamonas sp.ZF-3 in the bacterial sludge are not effectively reserved, only 1.59 percent of the water loss is detected with serious water change, and the method is the main reason for poor COD degradation effect, especially for the degradation of phenolic compounds and heterocyclic compounds.

The ratio of Firmicutes (1.69%) and Bacteroidetes (13.96%) in the carriers is relatively increased, probably because the anaerobic and anoxic environment provided inside the carriers promotes the growth of the corresponding anaerobic and facultative bacteria. The microorganisms commonly exist in wastewater treatment, the Firmicutes bacteria of Firmicutes can produce extracellular enzymes such as cellulose, lipase, protease and the like, and are closely related to the hydrolysis and utilization of refractory organic matters in coking wastewater, while the bacteroides plays an important role in denitrification and effective degradation of refractory macromolecular organic matters. In the case of nitrifying bacteria, Nitrospira (1.90%) was detected in the vector, and its relative abundance increased from 0.72% before the vector reaction to 1.90% after 123 days of the vector reaction. These increments represent an enhancement in ammonia nitrogen removal capability, which correlates with NH during operation₄ ⁺The variation of-N removal amounts was consistent. However, due to the defect of autotrophic Nitrospira in the activated sludge, the further improvement of the ammonia nitrogen degradation capability needs to consider the introduction of additional Nitrospira during the preparation of the carrier. Because a large amount of toxic organic matters in water are not fully degraded, nitrobacteria in the bacterial sludge are inhibited and consumed in the sludge, so the bacterial sludge shows poor denitrification effect.

3.5 functional differential analysis of vector microbial communities

The gene function composition of the genome of the sequenced microorganism in the immobilized carrier and bacterial sludge microflora is further analyzed by functional analysis software PICRUSt. Results as shown in fig. 16, there were significant differences (p <0.05) in functional abundance of the immobilized carrier and sludge microbial community functions such as cell Membrane Transport (Membrane Transport), xenobiotic biodegradation and Metabolism (xenobiotic Metabolism), Energy Metabolism (Energy Metabolism), and Replication and Repair (Replication and Repair, 7.1%), wherein the immobilized microbial carrier population showed higher abundance in Membrane Transport and xenobiotic biodegradation metabolic functions, indicating its good degradation in the biodegradation of the coking wastewater. The bacterial sludge community shows higher abundance energy metabolism and replication and restoration functions, which indicates that the activity of microorganisms in the bacterial sludge is inhibited to a certain extent, and the microorganisms need to carry out more basic metabolism to adapt to the high-load polluted environment and carry out cell restoration.

The above-described embodiments are merely illustrative of the preferred embodiments of the present invention, and do not limit the scope of the present invention, and various modifications and improvements of the technical solutions of the present invention can be made by those skilled in the art without departing from the spirit of the present invention, and the technical solutions of the present invention are within the scope of the present invention defined by the claims.

Claims

1. A preparation method of an immobilized microorganism carrier is characterized by comprising the following steps:

2. Root of herbaceous plantThe method according to claim 1, wherein in step (1), the polyvinyl alcohol, the sodium alginate, and the CaCO are added₃And the mass volume ratio of the deionized water is10 g: 2 g: 1 g: 90 mL.

3. The method according to claim 1, wherein in the step (1), the heating dissolution is heating dissolution at 90 ℃ for 20 min.

4. The method according to claim 1, wherein in step (3), the microorganism of the microorganism Comamonas sp.ZF-3 is prepared by: inoculating the high-efficiency bacterium Commamonas sp.ZF-3 into an LB culture medium, and culturing for 16h under the conditions of pH 7.0, 30 ℃ and 130r/min to obtain a fermentation broth of the high-efficiency bacterium Commamonas sp.ZF-3; centrifuging the fermentation liquor, and removing the supernatant to obtain a precipitate; and washing the precipitate by phosphate buffer solution, centrifuging again, and taking the precipitate to obtain the high-efficiency bacterium Comamonas sp.ZF-3 fermentation thallus.

5. The preparation method according to claim 1, wherein in the step (3), the mass ratio of the zymophyte of the high efficiency bacterium Comamonas sp.ZF-3 to the activated sludge is 1: 4.

6. the preparation method according to claim 1, wherein in the step (3), the bioaugmentation sludge composed of the zymophyte of the bacterium Commamonas sp.ZF-3 and the activated sludge is added into the immobilized microorganism carrier in an amount of 28 mg/mL.

7. The preparation method according to claim 1, wherein in the step (4), the cyclic freeze-thaw shaping is cyclic freeze-thaw for 4 times, and each freeze-thaw is specifically: the mixture was frozen at-8 ℃ for 20h and then thawed at room temperature for 4 h.

8. The method according to claim 1, wherein in the step (5), the chemical crosslinking is carried out for 70 min.

9. An immobilized microorganism carrier prepared by the preparation method according to any one of claims 1 to 8.

10. Use of an immobilized microorganism carrier according to claim 9 in the treatment of coking wastewater.